Successful protein folding involves the conversion of a linear polypeptide into a stable and biologically active 3D structure. This process has been studied for decades by denaturing purified, full-length polypeptides in vitro, diluting away the denaturant, and observing the refolding process. Careful measurements of refolding kinetics and populated partially folded conformations have revealed much about the refolding process. What is still lacking, however, is an understanding of how these in vitro results relate to protein folding in vivo. Protein folding in vivo is a fundamentally different process: In particular, the starting ensemble for folding in vivo is a growing nascent polypeptide chain, rather than a full-length chain. We have very little information about how this fundamentally different landscape for the growing chain relates to refolding mechanisms observed in vitro. Yet this difference may help explain why there are some native state topologies that are well represented in vivo, but difficult to refold in vitro. In this proposal, the influence of translation on protein folding in vivo will be studied from two perspectives: (1) The effects of translation pausing on folding yield. Rare codons can cause the ribosome to pause during translation, and literature reports have connected pauses with folding yield. For this proposal, novel methods have been developed to identify and modulate rare codon-derived translation pauses, and the effects on downstream folding. (2) The relationships between co-translational nascent chain conformations, in vitro refolding conformations, and the conformations of free, truncated polypeptides. Does protein refolding in vitro faithfully reproduce the conformations of nascent chains? For native structures, with contacts between distant portions of the polypeptide chain, this is unlikely. What alternative conformations are formed by these nascent chains? Are they also formed by free peptides? Results from in vitro refolding studies have revealed general principles for protein refolding;results from this proposal will be used to develop principles for protein folding in vivo.